1. Field of the Invention
The invention relates to assays for deletion of human genes which may be involved in neoplastic disorders. More particularly, the invention relates to the efficient detection of the deficiency of the human glutathione transferase (GST) isoenzyme GST-mu through the analysis of human blood or tissue samples in the clinical laboratory, as a means of predicting susceptibility to human neoplasia, including lung cancer. The invention further relates to means for predicting resistance of tumors to certain anticancer drug therapy as well as to the identification of individuals who may be at abnormally high risk of toxic effects upon exposure to chemicals which are detoxified by the GST-mu protein.
2. Summary of the Related Art
Mullis et al., U.S. Pat. No. 4,683,202 discloses the polymerase chain reaction (PCR).
Saiki et al., (1988, Science 239: 487-491) discloses the use of the thermostable Taq polymerase in PCR.
Kim and Smithies (1988, Nucleic Acids Res. 16: 8887-8903) discloses conditions for carrying out PCR on cell lysates in a recombinant fragment assay.
Boehm (1989 Clin. Chem. 35: 1843-1848) discusses the use of PCR for prenatal testing for inherited disorders.
Lynas et al., (1989, J. Gen. Virol. 70: 2345-55) discloses the use of PCR in the detection of viral gene expression.
Losekoot et al., (1989, Hum. Genet. 83: 75-78) discloses detection of a franeshift insertion mutation in a thalassemia gene, using PCR.
Boerwinkle et al. (1989, Nucleic Acids Res. 17: 4003) discusses the use of PCR to type an insertion/deletion polymorphism in a human apolipoprotein B gene.
Shvarts et al., (1989, Biorg. Khim. 15: 556-559) discloses characterization of the molecular nature of a deletion in a beta-globin gene, using PCR.
Xu et al., (1989, Teratogenesis Carcinog. Mutagen. 9: 177-187) discloses the use of PCR for deletion screening at the hgprt locus in cultured Chinese hamster cells.
Wrischnik et al., (1987, Nucleic Acid Res. 15: 529-542) discloses the identification of length mutations in human mitochondrial DNA by direct sequencing of PCR-amplified DNA.
While the related art discloses a variety of uses for PCR in the identification of mutations, there is no teaching of the use of PCR to detect large deletions in genes contained in highly necrotic tissues. Additionally, the existing art does not provide a means for effectively carrying out PCR at the GST-1 locus, and the existing technology for detecting the absence of GST-mu protein (MUKIT, Medlabs, Dublin, Ireland) relies on immunological methods to detect protein, and is thus unsuited to the assay of tissue samples, particularly highly necrotic tissue samples, such as those found in tumors.
There is a need for a better assay for GST-1 presence and expression, because of the significant role of the GST-mu isozyme in human physiology.
Seidegard et. al., (1985, Carcinogenesis 6: 1211-1216) teaches that the human glutathione transferase GST-mu is frequently deficient in individuals, and that such deficiency can result in altered metabolic activity toward certain toxic chemicals.
Seidegard et al. (1988, Proc. Natl. Acad. Sci. USA 85: 7293-7297) teaches that GST-mu deficiency is due to homozygous deletion of the GST-1 gene; and further provides DNA sequence information for the human cDNA for GST-mu.
Seidegard et al., (1986, Carcinogenesis 7: 751-753) teaches that GST-mu deficiency is associated with an increased risk for lung cancer.
Lai et al., (1988, J. Biol. Chem. 263: 11389-11395) discloses sequence information for a rat genomic clone related to the human GST-1 gene.